Project Summary Diabetes-reduced cardiac vagal activity is involved in sudden cardiac death and is responsible for high mortality in diabetic patients. Increasing cardiac vagal tone significantly limits cardiac dysfunction and reduces mortality. However, the potential mechanisms involved in reduced cardiac vagal activity in type 2 diabetes mellitus (T2DM) are poorly understood. Cardiac vagal ganglionic neurons (a final common pathway for vagal control of cardiac function) regulate acetylcholine release to influence cardiac function. Ca++ influx through voltage-gated Ca++ channels is a key trigger for acetylcholine release from these neuronal terminals. Our recent study has shown that expression and current density of N-type Ca++ channels in cardiac vagal ganglionic neurons are decreased in T2DM rats. Rat cardiac vagal ganglia are divided into the sinoatrial ganglion and the atrioventricular ganglion (AVG). The ventricular myocardium only receives the projection of nerve terminals from AVG neurons. Based on our preliminary data, we hypothesize that T2DM-mediated hydrogen peroxide (H2O2) overproduction in AVG neurons inhibits N-type Ca++ channel function via repressor element 1-silencing transcription factor (REST) signaling and/or by direct action, which further contributes to attenuation of ventricular vagal neuronal function in T2DM. Using multi-faceted technical approaches ranging from whole-animals to cellular-molecular levels, we will design in vivo and in vitro studies in sham and T2DM rats to assess these questions. In Specific Aim 1, we will address if T2DM induces ventricular vagal neuronal dysfunction as measured by N-type Ca++ channel expression and activation, cell excitability, and intracellular Ca++ levels in ventricular vagal neurons, as well as ventricular acetylcholine release from vagal nerve terminals. In Specific Aim 2, we will test how H2O2 overproduction modulates function of AVG neurons in T2DM through REST signaling. In Specific Aim 3, we will determine if impairment of ventricular vagal neurons contributes to ventricular electrical and contractile dysfunction in T2DM. These studies will further our understanding of the cellular and molecular mechanisms responsible for impaired cardiac vagal activity in T2DM and will also explore potential therapeutics for improving cardiac vagal activity and reducing mortality in T2DM.